1. Field of the Invention
This invention pertains generally to the field of cytometry and more particularly to cytometry systems and methods that provide real time three dimensional image enhancement, such as segmentation, contrast, and edge sharpening in response to images gathered by high-speed confocal microscopy that may be based on surface tracking.
2. Description of Related Art
Recent microscopic techniques and systems have provided practical cytographic imaging and segmentation in two dimensions. The fully automated scanning of cells using these microscopic techniques, such as performed on an operator independent cytometer, has been found to yield important diagnostic and research information for a number of biomedical applications, in particular those involving tissue research. For example, in the case of fluorescent cytology the use of digital light processing techniques with a cytometer can yield images of cell nuclei stained with a fluorescent dye which can be analyzed to extract relevant information, such as quantities of DNA, nuclear sizes, shapes and positions.
In addition, the ability to segment objects from the background and from image artifacts has shown itself to be an important capability when studying tissues. As utilized herein the term “segmentation” refers to partitioning an image into parts (“segments”) that may be individually processed. In the context of tissue research the segments of interest, which may also be referred to as “objects”, are typically individual cells. The use of segmentation reduces the vagaries and overhead associated with human interpretations while increasing the speed, efficiency, and repeatability with which results are produced.
Once segmented, the binary image may be analyzed for size and shape information and overlaid on the original image to produce integrated intensity and pattern information. In many applications it is advantageous to process a large number of cells (i.e. 104 to 106 cells), because of the inherent biologic variability. By way of example, performing an analysis spanning a large numbers of cells is particularly important in PAP smear screening, (and tests having a similarly large sample size) in which all cells on the slide may be measured to provide an accurate analysis that is not subject to false negative diagnoses. It should be appreciated that at this level of detail analytical intervention by a human operator is generally impractical. Accurate analysis in this and other situations involving a large number of objects requires the use of operator independent automation for the thousands of images collected for each slide.
Scanning two-dimensional cytometry has therefore become a successful commercial tool for tissue engineering. Representative of these systems are the teachings found in U.S. Pat. Nos. 5,995,143; 5,790,710; 5,790,692; and 5,548,661; and publication US2002/0186874 along with US International publication WO 01/54061 which are included herein by reference. Two-dimensional cytometry techniques typically utilize a form of confocal microscopy utilizing a micromirror array, such as taught within U.S. Pat. Nos. 5,587,832 and 5,923,466 whose teachings are also included herein by reference. A multiparallel three dimensional optical microscopy system is described by J. Price in publication number US2002/0001089 which is included herein by reference.
However, it should be appreciated that relying on two-dimensional images of natively three-dimensional (3D) structures can lead to errors and mis-estimations. Obtaining 3D images, however, is generally impractical for most applications as the current methods are slow and subject to various errors.
FIG. 1 represents a conventional 3D confocal scanning technique in which vertical stacks of optical sections are collected by changing focus on the microscope. After collection of the Z-scan (images collected along the Z-direction), lateral stage motion (X and Y) is performed to move from capturing one Z-direction stack to the next. The length of time required for performing this step and repeat process on a sample is generally prohibitive for most applications. The resultant set of collected data forms an XYZ image montage from which an extended 3D image may be rendered. In a typical confocal microscope used for collecting 3D images, the user interactively sets the top and bottom limits of the Z-scan and a computer-controlled system collects optical sections using a predetermined focus increment.
Current method and system for collecting 3D images are impractically slow for most tissue study applications. Confocal microscopes are currently too slow while three-dimensional (3D) imaging tools lack the accuracy and features required for scientists and tissue engineers to analyze from millions to billions of cells in tissue volumes It should be appreciated, for example, that blood has approximately five billion cells per milliliter (5 B cells/ml).
Therefore, a need exists for a 3D imaging that allows rapid autonomous imaging while still providing image segmentation as outlined above for tissue research and clinical applications. The present invention satisfies those needs, as well as others, and overcomes the deficiencies of previously developed 3D imaging solutions.